DESCRIPTION: We have recently discovered that human lactoferrin, a member of the lactotransferrin family of iron binding proteins, has proteolytic activity. Lactoferrin is one of the most abundant proteins in milk and most other human secretions, and is likely to contribute to the innate immune defense mechanisms on mucosal surfaces. It was unexpected that such a widely studied protein is a protease, but this has now been confirmed using recombinant lactoferrin expressed in baby hamster kidney cells, Aspergillus awamori, and baculovirus expression systems. Bovine milk lactoferrin has similar activity. We have shown that human lactoferrin is a serine-type protease, and the enzyme active site is a serine-lysine dyad located in the amino-terminal, globular N-lobe of the protein. In addition, we have identified the peptide bonds that lactoferrin cleaves in the Haemophilus influenzae Hap adhesin and IgA protease precursor, two virulence proteins that are secreted to the outer membrane by the autotransporter pathway. Lactoferrin also cleaves proteins from the outer membranes of the dental pathogens Actinobacillus actinomycetemcomitans and Porphyromonas gingivalis, and from Shigella enteric pathogens. Among the known substrates are adhesins that mediate bacterial attachment to host epithelial cells, and lactoferrin cleavage reduces bacterial adhesiveness, in vitro. In our proposed studies we will develop quantitative assays for lactoferrin enzyme activity. These will be based on fusion proteins that are designed to incorporate lactoferrin-susceptible polypeptides whose cleavage releases an easily measurable reporter. Short substrates will be introduced into immobilized cellulase of Cellulomonas fimi, allowing estimation of lactoferrin activity by measuring released cellulase activity. Longer substrates will be expressed in E. coli as fusion proteins that have bacterial alkaline phosphatase as the reporter. This strategy will then be used to identify the peptide bonds cleaved by lactoferrin in Aae, an autotransporter adhesin protein of the periodontal pathogen Actinobacillus actinomycetemcomitans. Finally, we will explore the role of lactoferrin oligosaccharides in modifying proteolytic activity. This will be by site-specific mutations of one or more asparagine glycan attachment sites in recombinant lactoferrin expressed in insect cells. These basic studies will address the long-term goal of producing proteolytically active, recombinant lactoferrin for clinical control of bacterial attachment, colonization, and infection of oral and other mucosal surfaces.